Molecular Mechanisms and Design of Protein Folding, Function, and Aggregation

蛋白质是精巧的分子机器——它们发挥着广泛的功能，从充当催化剂，到专门识别和结合各种分子，再到结构支持作用。蛋白质的初级氨基酸序列决定了它如何折叠成三维结构，而三维结构又决定了它的功能。尽管进行了广泛的研究，但控制蛋白质折叠、聚集和功能的分子机制的细节仍然不清楚。确定这些机制非常重要，因为这些知识最终将使我们能够精确预测和控制任何蛋白质的特定特性：在自然过程中，在疾病中，在生物技术中。它将使我们能够为任何所需的应用设计或工程蛋白质！天然和工程蛋白广泛用于研究和工业，并通过其在材料科学中的应用，以及作为催化剂和药物，正在改变生物技术。蛋白质序列、结构和功能知识的爆炸性增长，以及蛋白质的计算建模，为揭示决定蛋白质特性的分子机制奠定了基础。例如，基于我们对蛋白质溶解度的实验测量，我们最近开发了新的基于结构的方法来预测和设计被称为Adnectins的工程靶结合蛋白的溶解度。对于许多蛋白质，Adnectins在细菌中表达时形成不溶性包涵体（insoluble inclusion body, IBs）， IB形成的水平与纯蛋白的溶解度有关。我们对Adnectins的发现对于在开发用于医疗应用的Adnectins时控制溶解度很重要。此外，我们还提出了一种利用淬火H/D交换核磁共振光谱（NMR）对IBs进行高分辨率结构分析的方法。该方法揭示了IBs的类天然结构，对IBs作为功能材料的开发具有重要意义。在另一个项目中，我们开发了一种核磁共振和分子动力学相结合的计算方法，在原子分辨率上揭示了Hisactophilin中ph依赖性开关功能的机制，该功能控制细胞运动。这种强大的方法广泛适用于定义局部结构稳定性和控制蛋白质结构和功能的替代状态的种群。我们对这些蛋白质和其他蛋白质的发现（包括我们设计的许多突变体，以系统地增加我们的知识）是使用一系列实验（光谱学，量热法，光散射）和计算工具获得的。我们将在之前令人兴奋的发现的基础上，推进对蛋白质如何工作以及如何利用蛋白质进行无数应用的认识；这项研究将为许多高素质的蛋白质开发人才提供有价值的跨学科合作和国际培训，以进一步造福社会。

Proteins are exquisite molecular machines - they perform a vast range of functions, from acting as catalysts, to specifically recognizing and binding all kinds of molecules, to structural support roles. The primary amino acid sequence of a protein determines how it folds to its 3D structure, which in turn determines its function.  Despite extensive study, the details of the molecular mechanisms governing protein folding, aggregation and function remain obscure. Defining these mechanisms is of tremendous importance because such knowledge will ultimately enable the precise prediction and control of the specific properties of any protein: in natural processes, in disease and in biotechnology. It will give us the ability to engineer or design proteins for any desired application! Natural and engineered proteins are widely used in research and industry and are transforming biotechnology through their applications in material science, and as catalysts and drugs. Explosive growth in knowledge of protein sequences, structures and functions and in computational modeling of proteins has set the stage for uncovering the molecular mechanisms that determine protein properties. For example, based on our experimental measurements of protein solubility, we recently developed new structure-based methods to predict and design the solubility of engineered target binding proteins known as Adnectins. As for many proteins, Adnectins form insoluble inclusion bodies (IBs) when expressed in bacteria, and the level of IB formation is related to the solubility of the pure protein. Our findings for Adnectins are important for controlling solubility when developing Adnectins for medical applications. In addition, we advanced a method for high resolution structure analysis of IBs using quenched H/D exchange nuclear magnetic resonance spectroscopy (NMR). Applying this method revealed native-like structure in IBs, which has important implications for developing IBs as functional materials. In a separate project, we developed a combined NMR and molecular dynamics computational method that revealed at atomic resolution the mechanism of pH-dependent switching function in Hisactophilin, which controls cell movement. This powerful method is widely applicable to defining local structural stability and the population of alternative states that control protein structure and function. Our discoveries for these and other proteins (including numerous mutants which we design to systematically grow our knowledge) are obtained using a battery of experimental (spectroscopy, calorimetry, light scattering) and computational tools.  We will build on our previous exciting discoveries to advance knowledge of how proteins work and how they can be harnessed for a myriad of applications; this research will provide valuable collaborative interdisciplinary and international training for many highly qualified personnel in developing proteins for further great benefit to society.